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Promoter-trap identification of wheat seed extract-induced genes in the plant-growth-promoting rhizobacterium Azospirillum brasilense Sp245

¹ Université de Lyon, Lyon, F-69003, France
² Université Lyon 1, Lyon, F-69003, France
³ CNRS, UMR 5557, Ecologie Microbienne, Villeurbanne, F-69622, France
⁴ CNRS, UMR 5534, Centre de Génétique Moléculaire et Cellulaire, Villeurbanne, F-69622, France

Correspondence
Claire Prigent-Combaret
prigent{at}biomserv.univ-lyon1.fr

Azospirillum strains have been used as plant-growth-promoting rhizobacteria (PGPR) of cereal crops, but their adaptation to the root remains poorly understood. Here, we used a global approach based on differential fluorescence induction (DFI) promoter trapping to identify genes of the wheat isolate Azospirillum brasilense Sp245 that are induced in the presence of spring wheat seed extracts. Fluorescence-based flow cytometry sorting of Sp245 cells was validated using PlacZ, PsbpA and PnifH promoters and egfp. A random promoter library was constructed by cloning 1–3 kb Sp245 fragments upstream of a promoterless version of egfp in the promoter-trap plasmid pOT1e (genome coverage estimated at threefold). Exposure to spring wheat seed extracts obtained using a methanol solution led to the detection of 300 induced DFI clones, and upregulation by seed extracts was confirmed in vitro for 46 clones. Sequencing of 21 clones enabled identification of seven promoter regions. Five of them displayed upregulation once inoculated onto spring wheat seedlings. Their downstream sequence was similar to (i) a predicted transcriptional regulator, (ii) a serine/threonine protein kinase, (iii) two conserved hypothetical proteins, or (iv) the copper-containing dissimilatory nitrite reductase NirK. Two of them were also upregulated when inoculated on winter wheat and pea but not on maize, whereas the three others (including PnirK) were upregulated on the three hosts. The amounts of nitrate and/or nitrite present in spring wheat seed extracts were sufficient for PnirK upregulation. Overall, DFI promoter trapping was useful to reveal Azospirillum genes involved in the interaction with the plant.

The GenBank/EMBL/DDBJ accession numbers for the sequence data obtained in this work are given in Table 3 and Supplementary Table S1.

A supplementary table of spring wheat seed extract-inducible cryptic fusions identified by DFI in A. brasilense Sp245 is available with the online version of this paper.

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